Demyelination and Multiple Sclerosis.
Demyelination is a neuropathological state where the insulating myelin sheath on the axons of the neurons is degraded, the pathogenesis of which could be due to a variety of causes.1 Multiple sclerosis (MS), one such clinical condition, is a chronic and most common demyelinating disease, affecting about 2.2 million people worldwide.2 It is characterized by a patchy degradation of myelin on the axons, known as demyelinated lesions, and the healing of these patches occurs via scar formation called plaques.
A variety of causes such as genetic, immunological and environmental factors are suggested to play a role leading to this condition.3 The most common theory is the autoimmune theory which postulates that sensitization of T cells in the periphery leads to their travel through a disrupted blood brain barrier to attack and destroy myelin.4 Several CNS proteins have been shown to induce this condition, including myelin proteolipid protein (PLP),5 myelin-associated glycoprotein (MAG),6 myelin oligodendrocyte glycoprotein (MOG),7 transaldolase and S100.6 Genetic studies indicate the involvement of about 30 single nucleotide polymorphisms (SNPs), although it remains to be seen as to the relevance of these SNPs for MS therapeutics development.8 
Current MS therapies reduce the frequency of relapses but do not delay the progression of the disease nor do they reverse the destruction of myelin.9,10,11 The most popular treatment is Copaxone™ (also known as Copolymer-1, Cop-1, or Glatiramer acetate), marketed by Teva Pharmaceuticals. This is an immunomodulator drug and is a random polymer of four amino acids-glutamic acid, lysine, alanine and tyrosine—in the same proportion found in myelin basic protein (MBP).
Currently, there is a need for novel mechanisms of preventing and potentially reversing demyelination, such that the treatment options for demyelinating diseases such as multiple sclerosis can be conceived with better safety profiles and with clear molecular mechanisms of action.
Citrullination and Demyelination.
In general, immunological self-tolerance is an important defense against many autoimmune diseases and its breakdown in the body leads to various autoimmune diseases. This primarily arises from the immune recognition of self-proteins that have undergone post-translational modifications under pathophysiological conditions that would not happen under normal circumstances.
Citrullination, a post-translational event, in general is involved in many cellular processes such as gene regulation, embryonic development and differentiation.12,13 Lately, the abnormal role of (hyper)citrullination in a variety of diseases has been uncovered, including in MS, rheumatoid arthritis, Alzheimer's, scrapie, psoriasis and Creutzfeld-Jacob disease.14,15 
In MS, extensive studies of hypercitrullinated MBP indicated that MBP, a key component of the myelin sheath and critical for the maintenance of myelin compaction, contained the non-coded amino acid citrulline in abnormal proportions. In normal brain, the “citrullinated MBP” accounts for 20% of the total MBP, whereas in chronic MS it accounts for 45%16 and in fulminating MS it is 90% of the MBP.17 In a number of studies using a variety of biophysical techniques,18,19,20,21,22 it was demonstrated that citrullinated MBP prevented compaction of the bilayer, resulting in destabilization of the membrane and subsequent degradation leading to demyelination, and an irreversible damage to the axons.23,24 
Thus, hypercitrullination is at the root of neuropathogenesis due to demyelination. In the central nervous system, peptidyl arginine deiminases (specifically PAD2 and PAD4) are responsible for the citrullination.
PAD Enzymes and Citrullination.
Peptidyl arginine deiminase (PAD) catalyzes the post-translational citrullination of proteins.25,26,27 Citrullination is the process of deimination of Arg residues on select proteins, or in other words, transformation of Arg into citrulline via deimination (Scheme 1). There are five isozymes of PAD that exist in humans: PAD-1, -2, -3, -4 and -6. Their expression in tissues varies significantly, regulated by transcriptional and post-transcriptional mechanisms. PAD2 and PAD4 are specifically implied in MS, as enhanced levels of these two isoforms are observed in CNS under inflamed conditions.28,29

There is convincing evidence in vivo that higher levels of PAD activities and hypercitrullination are observed in MS.30 For example, a routinely used MOG-EAE model for MS, which is a CD4(+) T cell-driven model, induced with the immunodominant 35-55 peptide of myelin oligodendrocyte glycoprotein (pMOG35-55) was used to test whether citrullination of a T cell epitope can contribute to disease etiopathology.23,31 In this experimental model, the PAD2 and PAD4 enzymes were significantly upregulated in the inflamed CNS of the animals. T cells that responded specifically to the citrullinated pMOG could not initiate the EAE lesion, but these cells could provoke exacerbation of pathology if transferred into mice with an ongoing EAE. This experiment strongly suggested that once inflammation in MS is established, citrullination of target autoantigens can allow an expanded repertoire of T cells to contribute to CNS pathology, and enhanced levels of PAD enzymes are observed in these tissues.31 A similar study using the peptides from myelin basic protein (MBP) epitopes indicated that self-antigens could potentially trigger the disease in susceptible individuals carrying citrullinated peptide epitopes.32,33 
Inhibitors of PAD Enzymes:
A non-specific, active site PAD inhibitor, 2-chloroacetamidine (2CA), attenuated MS disease, decreased the amount of citrullinated protein and decreased PAD activity in the brain in four animal models of MS: two neurodegenerative and two autoimmune disease models.34 
2CA is a covalent inhibitor of PAD4 (FIG. 2).
A non-specific, active site PAD inhibitor, 2-chloroacetamidine (2CA), was previously shown to attenuate MS disease, decrease the amount of citrullinated protein and decrease PAD activity in the brain in four different animal models of MS.35 In one of these murine models, early and prolonged 2CA administration essentially prevented the disease. However, fully progressive clinical disease re-emerged promptly after therapy cessation at 6 months.34 Thus, there remains a need to identify improved therapeutics for inhibiting disease-related hypercitrullination.
Structures of PAD Enzymes.
Structurally, PAD enzymes are Ca2+-dependent enzymes that catalyze the conversion of arginine residues in proteins to citrulline via the deimination of the guanidinium moiety in the side chain of Arg residues.36,37 The structure consists of the N-terminal domain predominately folded into β-sheets, and the C-terminal domain where the catalytic site is located. The catalytic site, where the substrate binds, has two Asp residues, one His residue and a Cys residue that are involved in the deimination reaction. Acidic amino acids, Asp350 and Asp473, function as general base residues during the hydrolysis of the amine in the guanidinium moiety of the peptidyl arginines. These two Asp residues are located in the bottom of the substrate-binding pocket (FIG. 2). 2CA, due to its acetamidine structure carrying a positive charge, binds at this anionic pocket and modifies the Cys residue that is in close proximity (FIG. 1). 2CA does not carry any additional structural features that provide it with specificity to inhibit PAD enzymes only, and not any other similar enzymes.
Over the past decade, there have been only a handful of efforts focused on understanding various ligands, their interactions and the inhibitors targeting PAD enzymes, and most notably, various peptide derivatives to understand the substrate and inhibitor properties targeting PAD enzymes.33,38,39,40 The most potent non-peptidic compounds from these investigations are chlortetracycline, a tetracycline derivative with an IC50 of 100+10 μM as a competitive inhibitor and a substrate analog, F-amidine with an IC50 of 21±2.1 μM as an irreversible inactivator.
Non-immune small molecule therapies targeting specific neurological mechanisms are much needed for neurodegenerative diseases because immune-based therapies are not effective. PAD enzymes hypercitrullinate proteins in the brain leading to the pathology and neurodegeneration in Alzheimer's disease (AD).41 This mechanism has been shown to operate in other neurodegenerative diseases such as MS, Parkinson's disease, amyotropic lateral sclerosis (ALS), multiple systems atrophy (MSA) and prion diseases such as Creutzfeldt-Jakob disease where hypercitrullination of myelin, higher expression of PAD2 and PAD4, and associated neurodegeneration are also observed.58,42,43,44,45,46,47,48,49,67 PAD2 and PAD4 are shown to cause the pathology in brain regions such as hippocampus and cerebral cortex, ultimately leading to neurodegeneration and cell death. In MS and Alzheimer's disease, enhanced levels and activities of PAD, hypercitrullinated proteins, and ultimately neurodegeneration have been correlated with disease in patients' brain samples. In MS animal models, inhibition of PAD activities by treatment with small molecule inhibitors leads to the prevention and reversal of neurodegeneration and/or highly reduced inflammatory response in the brain.50,51 
PAD enzymes and their catalytic activities leading to higher levels of citrullinated proteins in brain regions such as hippocampus, cerebral cortex and myelin are correlated to neurodegenerative changes typical for AD.52 Throughout the process of neurodegeneration process, PAD2 and PAD4 specifically are abundantly expressed in brain.69,53 Collectively evidence suggests that an abnormal activation of PAD2 and PAD4 in hippocampi of patients with AD finally leading to neurodegeneration.70 Similar observations have been made in the mouse and rat models of AD.54 Observations described here in the area of protein citrullination, inhibition of PAD enzymes and the development of novel inhibitors of PAD to prevent and/or reverse neurodegeneration and associated inflammation67,68 will have applications as novel therapeutics targeting neurodegenerative diseases such as AD, MS, ALS, MSA, CJD etc.
Though current literature suggests the mammalian enzyme peptidylarginine deiminase type II (PAD2) does not use free arginine as a substrate55 the enzyme is responsible for the conversion of peptide-bound positive arginine to neutral citrulline by means of a calcium (Ca2+)-induced deimination reaction.55,56 This deimination affects the behaviour of proteins in the cellular environment since it induces proteins to unfold, which could subsequently act as a catalyst for the aggregation of susceptible proteins.57 The reaction catalyzed by PAD2 is localized to peptides in the astrocytes in the cerebral tissue that, as mentioned above, are reservoirs of arginine storage within the brain.58 